The characterization of ESBL genes in escherichia coli and klebsiella pneumoniae causing nosocomial infections in vietnam

The characterization of ESBL genes in Escherichia coli and Klebsiella Nguyen Hoang Thu Trang1, Tran Vu Thieu Nga2, James I Campbell2,3, Nguyen Trong Hiep1, Jeremy Farrar2,3, Stephen Bake

The characterization of ESBL genes in Escherichia coli and Klebsiella

Nguyen Hoang Thu Trang1, Tran Vu Thieu Nga2, James I Campbell2,3, Nguyen Trong Hiep1, Jeremy Farrar2,3, Stephen Baker2,3, Pham Thanh Duy2

1

Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Medicine and Pharmacy, Ho Chi Minh City, Vietnam

2

The Hospital for Tropical Diseases, Wellcome Trust Major Overseas Programme, Oxford University Clinical Research Unit, Ho Chi Minh City, Vietnam

3

Centre for Tropical Medicine, Oxford University, Oxford, United Kingdom

Abstract

Background: Extended-spectrum β-lactamases (ESBLs) are enzymes capable of hydrolyzing oxyimino-β-lactams and inducing resistance to third generation cephalosporins The genes encoding ESBLs are widespread and generally located on highly transmissible resistance

plasmids We aimed to investigate the complement of ESBL genes in E coli and Klebsiella pneumoniae causing nosocomial infections in

hospitals in Ho Chi Minh City, Vietnam

Methodology: Thirty-two non-duplicate isolates of E coli and Klebsiella pneumoniae causing nosocomial infections, isolated between March and June 2010, were subjected to antimicrobial susceptibility testing All isolates were PCR-amplified to detect the blaSHV, blaTEM and

blaCTX-M ESBL genes and subjected to plasmid analysis

Results: We found that co-resistance to multiple antimicrobials was highly prevalent, and we report the predominance of the blaCTX-M-15 and

blaCTX-M-27 genes, located on highly transmissible plasmids ranging from 50 to 170 kb in size

Conclusions: Our study represents a snap shot of ESBL-producing enteric bacteria causing nosocomial infections in this setting We suggest

that antimicrobial resistance in nosocomial E coli and Klebsiella pneumoniae is rampant in Vietnam and ESBL organisms are widespread In

view of these data and the dramatic levels of antimicrobial resistance reported in Vietnam we advocate an urgent review of antimicrobial use

in the Vietnamese healthcare system

Key words:Enterobacteriacea; Extended-spectrum beta-lactamases; ESBL-encoding genes; Plasmid-mediated resistance; antimicrobials

J Infect Dev Ctries 2013; 7(12):922-928 doi:10.3855/jidc.2938

(Received 21 August 2012 – Accepted 08 November 2012)

Copyright © 2013 Trang et al This is an open-access article distributed under the Creative Commons Attribution License, which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited

Introduction

The production of β-lactamases is the most

common mechanism of bacterial resistance to the

β-lactam antimicrobials β-β-lactamase genes are

widespread and mutate in response to continuous

antimicrobial exposure This prolonged exposure has

led to the emergence of extended-spectrum

β-lactamases (ESBLs) [1] ESBLs are enzymes capable

of hydrolyzing oxyimino-β-lactams, such as third

generation cephalosporins, which include the

commonly used antimicrobials, ceftriaxone and

cefixime The dissemination of ESBLs is a global

problem, particularly in sentinel members of the

Enterobacteriaceae [2] Among the known ESBL

enzymes, the CTX-M-type β-lactamases, which

preferentially hydrolyze cefotaxime, were first

reported in the late 1980s and have undergone a rapid,

global spread The spread of CTX-M-type β-lactamases has been dramatic and greater than the impact of the TEM- and SHV-type ESBLs [3-6]

In Vietnam, the presence of pathogens expressing ESBLs has been increasingly reported over the past ten years A study conducted in 2001 in seven hospitals across Ho Chi Minh City in the south of Vietnam, found that 5.6 % of all Gram negative bacterial isolates were ESBL positive, with the rate of

positivity in Escherichia coli and Klebsiella pneumonia being 58 % and 23.6 %, respectively [7] A

further study, also conducted in Ho Chi Minh City, between February 2002 and May 2005, found that 33

% of all Gram-negative bacterial isolates were ESBL

positive From these ESBL positive isolates, E coli and K pneumoniae accounted for 74.1 % of all the

organisms isolated [8] A pan Asia-Pacific study

regarding Gram-negative bacilli from intra-abdominal

infections in 2007, found that the ESBL positivity rate

in Vietnam was 35.6 % (34.4 % and 39.1 % of the

ESBL positive strains were E coli and K pneumoniae,

respectively) [9] As such, the prevalence of ESBL

producing strains in Vietnam and the Asia-Pacific

region is now higher than those observed in Europe,

suggesting differing geographical pressures and

exposures to antimicrobials [9, 10]

ESBL producers can often transfer resistance to

multiple bacterial species through plasmid-mediated

conjugation [11] The widespread use of

antimicrobials, coupled with the transmissibility of

resistance determinants mediated by plasmids,

transposons, and integrons, contribute to increasing the

prevalence of antimicrobial resistance in pathogenic

members of the Enterobacteriaceae [12] These

elements pose serious problems in hospital settings

worldwide Therefore, surveillance of ESBLs

producing Enterobacteriaceae is necessary to add

insight into ESBL transmission, the emergence of

predominant ESBL groups and the mobile elements

inducing the dissemination of ESBL determinants In

Vietnam, limited studies have been performed

investigating the molecular characteristics of ESBL

genes and their corresponding mobile elements Here,

we aimed to define the characteristics of common

ESBL genes and their encoding plasmid profiles in

members of the Enterobacteriaceae causing

nosocomial infections in hospitalized patients in Ho

Chi Minh City, Vietnam

Methodology

Ethics statement

This study was conducted according to the

principles expressed in the Declaration of Helsinki and

was approved by the institutional ethical review

boards of the participating hospitals Samples were

collected as part of “standard of care” for treatment

and diagnosis; therefore, the institutional ethical

review boards did not require us to collect informed

consent

Clinical isolates, antimicrobial susceptibility testing

and ESBL phenotyping

The microbiology laboratories at Cho Ray and

Thong Nhat hospitals in Ho Chi Minh City isolated 72

bacterial isolates (E coli or K pneumoniae) causing

nosocomial infections demonstrating resistance to

ceftriazone and ceftazidime between March and June,

2010 Thirty-two of these isolates, comprising 23 E

coli and 9 K pneumonia, were latterly confirmed to be

ESBL producing at the laboratories of Oxford University Clinical Research Unit by the double-disc synergy test (the remainder were ESBL negative) [13] The double-disc synergy method utilizes discs containing cefotaxime (CTX) (30 µg) and ceftazidime (CAZ) only (30 µg) and both antimicrobials in combination with clavulanic acid (CLA) (10 µg) ESBL producing strains were identified as those with a greater than 5 mm increase in zone with the single antimicrobial compared to the combined antimicrobials All 32 bacterial isolates were additionally subjected to susceptibility testing by assessing the minimum inhibitory concentrations (MICs) against amoxicillin/ clavulanic acid (AMC), cefepime (FEP), ceftriaxone (CRO), imipenem (IPM), ciprofloxacin (CIP), nalidixic acid (NAL), trimethoprim/ sulfamethoxazole (SXT) and chloramphenicol (CHL) by E-test (AB Biodisk, Solna, Sweden) All antimicrobial susceptibility tests were performed on Mueller-Hinton agar and the resulting data were interpreted according to the Clinical and Laboratory Standards Institute guidelines [13]

Nucleic acid amplification and sequencing

Genomic DNA was isolated from all bacterial isolates from 1 mL of an overnight bacterial culture using the Wizard Genomic DNA Extraction Kit (Promega, Fitchburg, USA), according to manufacturer’s recommendations All isolates were

screened for the presence of blaSHV, blaTEM, blaCTX-M

ESBL genes using previously published primers [14,

15] Further characterization of the blaCTX-M was performed using the primers specific for CTX-M-1,

amplifications were performed using 30 cycles, of 30s

at 95oC, 30s at 57oC, and 90s at 72oC All PCR amplifications were performed using Taq DNA

concentrations of reagents (Bioline, London, UK) All PCR amplicons were sequenced using big dye terminators in a forward and reverse orientation on an ABI3130XL sequencing machine (ABI, Advanced Biotechnology Inc, Columbia, USA), according to the manufacturer’s recommendations Resulting DNA sequences were verified and aligned using BioEdit and Vector NTI Suite 7 software BLASTn at NCBI was used to compare all resulting ESBL gene sequences against additional ESBL sequences

The DNA sequences for blaTEM-1, blaCTX-M-1,

blaCTX-M-3,blaCTX-M-9, blaCTX-M-14, blaCTX-M-15, bla

CTX-M-27, blaCTX-M-55 genes the accession numbers J01749,

X92506.1, Y10278, AF174129.3, AF252622.2,

AY044436.1, AY156923.1 and DQ885477.1 were

downloaded from NCBI and aligned with the resulting

sequences

Plasmid extraction and visualization

Plasmid DNA was isolated from all ESBL

bacterial isolates using an adapted methodology

originally described by Kado and Liu [11] Briefly,

plasmid DNA was separated by agarose gel

electrophoresis in 0.7 % agarose gels with 1X TBE

buffer Agarose gels were subjected to 100V for 4

hours, stained with ethidium bromide and

photographed E.coli 39R861 containing plasmids of

7, 36, 63 and 147 kb was used for sizing plasmid

extractions on agarose gels [16] Plasmid DNA was

size-separated and analyzed using Bionumerics

software (Applied Maths, Sint-Martens-Latem,

Belgium)

Southern blotting and hybridization

Plasmid DNA was electrophoresed and transferred

to a Hybond N+ membrane (Amersham Biosciences,

Little Chalfont, UK) The PCR amplicons of blaTEM,

blaCTX-M-1, blaCTX-M-9 were labeled with horseradish

peroxidase using the ECL direct nucleic acid labeling

and detection systems kit (Amersham Biosciences, Little Chalfont, UK), and were used as hybridization probes Hybridization and detection were performed according to the manufacturer’s instructions

Bacterial conjugation

Conjugation was performed by combining equal volumes (500 µL) of overnight cultures grown in Luria-Bertani (LB) media of donor and recipient strains in 4 mL of sterile LB media The donor strains

were ESBL-producing isolates (E coli and K pneumoniae) and the recipient was a sodium azide resistant E coli (strain J53 resistant) Bacteria were

mixed in a 1:1 ratio at 37oC and incubated without agitation overnight Transconjugants were selected on

LB media containing sodium azide (100 µg/mL) and ceftriaxone (6 µg/mL) and were verified by plasmid extraction and visualization, as before Conjugation frequency was calculated by dividing the mean number of transconjugants by the mean number of recipient cells

Results

Antimicrobial susceptibility

All ESBL-producing isolates were resistant to ceftriaxone, of which 27/32 (84.3 %) isolates exhibited

an MIC of greater than 256 µg/ml Resistance to additional antimicrobials was common with 27/32 (84.3 %) resistant to ciprofloxacin, 28/32 (87.5 %)

Table 1 The antimicrobial resistance patterns of ESBL producing organisms

Bacterial isolates Number of antimicrobials resistant a Number of isolates Antimicrobial resistance phenotypes b

E coli (n = 23)

K pneumoniae (n=9)

a

From 8 tested, see methods

b AMC; amoxicillin/ clavulanic acid, FEP; cefepime, CRO; ceftriaxone, IPM; imipenem, CIP; ciprofloxacin, NAL; nalidixic acid, SXT; trimethoprim/ sulfamethoxazole and CHL; chloramphenicol

resistant to trimethoprim-sulfamethoxazole, 27/32

(84.3 %) resistant to nalidixic acid and 17/32 (53.1 %)

resistant to chloramphenicol (Table 1) More than 80

% of the isolates were resistant to between four and six

of the antimicrobials tested (Table 1)

Six out of 32 isolates (18.8 %) were resistant to the

fourth generation cephalosporin, cefepime, with an

additional 11/32 (34.4 %) demonstrating intermediate

resistance All ESBL-producing strains were sensitive

to the carbapenem, imipenem

Characterization of bla genes

PCR amplifications were performed to detect the

blaTEM, blaSHV and blaCTX-M genes The resulting

amplifications demonstrated that all 32 of the

ESBL-producing isolates carried a blaCTX-M gene, 24/32

isolates harbored an additional blaTEM gene and no

isolates carried a blaSHV gene All strains were additionally amplified with primers specific for the

three major CTX-M clusters, blaCTX-M-1, blaCTX-M-2 and

blaCTX-M-9 With these specific CTX-M cluster primers,

one E.coli isolate, produced an amplicon with both blaCTX-M-1 and blaCTX-M-9 primers, the remaining strains

produced single amplicons with either the blaCTX-M-1

primers or the blaCTX-M-9 primers and none tested

positive with the blaCTX-M-2 primers (Table 2) All 33 PCR amplicons were DNA-sequenced to identify the

specific blaCTX-M variants DNA sequence analysis

showed that multiple blaCTX-M loci were circulating in

the E coli and K pneumoniae isolates We identified one blaCTX-M-3, 15 blaCTX-M-15, two blaCTX-M-55, four

blaCTX-M-14 and 11 blaCTX-M-27 genes (Table 2) The

blaCTX-M-15 gene was the most predominant variant

(15/18 strains) among the blaCTX-M-1 cluster, and

Table 2 The characterization of ESBLgenes and their corresponding plasmids

Bacterial isolates CAZ zone size

(mm) ESBL gene(s) detected Size of ESBL Plasmid (Kb) a

Number of transferable plasmids

Maximum conjugation frequency b

E coli

K pneumoniae

a Estimated plasmid size by agarose electrophoresis with markers of known sizes

b Conjugation frequency per recipient cell

ND Not detected

blaCTX-M-27 (11/15 strains) was the most predominant

variant within the blaCTX-M-9 cluster Among the 15

blaCTX-M-15 carrying isolates, five were additionally

resistant to cefepime (MIC > 32 µg/ml) and seven

exhibited intermediate cefepime resistance (median

MIC = 16µg/ml)

Characterization of ESBL encoding plasmids

Plasmid profiling of 32 ESBL-positive isolates

demonstrated that all strains harbored at least one large

plasmid, ranging from 50 to 171 kb in size (Table 2)

Furthermore, the majority of the strains also harbored

multiple other plasmids, ranging from 1 to 50 kb in

size The number of plasmids in these isolates ranged

from one to nine and after gel sizing analysis, using a

binary scoring system with a Pearson correlation, we

found that plasmid profiles were not specific to E coli

or K pneumoniae (data not shown)

Plasmid DNA hybridization demonstrated that the

majority of the ESBL-producing strains (31/32 strains)

carried the bla genes on a large plasmid (ranging from

53.8 to 157 kb in size) (Table 2) These large plasmids

encoded a blaCTX-M only or both a blaCTX-M and a

blaTEM gene Among the 24 strains carrying both a

blaCTX-M and blaTEM genes, 18 strains carried these

genes on the same plasmid and four strains carried

these genes on different plasmids; we were unable to

confirm the PCR result for two strains as a presumed

consequence of plasmid instability after in vitro

passage (this was, however, latterly confirmed by

PCR) (Table 2) ESBL-producing strains containing

genes belonging to the blaCTX-M-9 gene cluster exhibited

less activity against ceftazidime in comparison to

strains carrying gene belonging to the blaCTX-M-1 gene

cluster Susceptibility testing against ceftazidime with

ESBL strains showed two distinct zone clearance areas

with the blaCTX-M-9 cluster (median; 18.3 mm) and the

blaCTX-M-1 cluster (median; 12.2 mm) (Table 2)

We performed bacterial conjugation experiments

on all 32 ESBL-producing strains (donors) using E

coli J53 as a recipient Results demonstrated that

plasmids harboring ESBL genes of twenty-two isolates

(69%) were transmissible via conjugation, with

conjugation frequencies ranging from 6.25 x 10-8 to 1

x 10-3 per recipient cell (Table 2) Of the 10 isolates

carrying non-transmissible plasmids, eight carried

ESBL plasmids with sizes greater than 100 kb and we

were unable to confirm the presence of ESBL genes

on plasmids by Southern Blotting in two (Table 2) We

further confirmed the transmission of ESBL plasmids

between both species (E coli to E coli) and genus (K pneumoniae to E coli)

Discussion

Our work shows that the CTX-M-type ESBLs are

the most common ESBL found amongst E coli and K

hospitals in Ho Chi Minh City Among the blaCTX-M

variants, blaCTX-M-15, blaCTX-M-14, blaCTX-M-27 were the

most common in E coli and in K pneumoniae, and blaCTX-M-15 accounted for 45 % of all blaCTX-M variants

A study regarding resistance gene characterization of

ESBL positive Shigella sonnei isolated at the Hospital

for Tropical Diseases in Ho Chi Minh City in 2009

found that blaCTX-M-15 was the most dominant blaCTX-M

variant, found in all but one ESBL positive Shigella sonnei [11] Our observations reflect the current rapid

and successful dissemination of CTX-M-type ESBLs

and the emergence of blaCTX-M-15 in Asia and globally

BlaCTX-M-15 first arose in India in 2000 and has become predominant globally within a decade [3, 10, 17, 18]

This particular blaCTX-M gene is generally found on large conjugative plasmids and is located downstream

of an ISEcp1 insertion sequence which explains its

remarkable transmission success [7, 11] The

CTX-M-15 type enzyme differs from that of CTX-M-3 type by

an asparagine to glycine substitution at codon 240, which leads to increased activity against ceftazidime These CTX-M-15 type enzymes may have been selected by the increasing use of ceftazidime in clinical practice [19-21]

We can additionally show that the ESBL-producing organisms additionally exhibited co-resistance against multiple antimicrobials from other classes Many studies have also reported co-resistance

to tetracycline, aminoglycosides, and fluoroquinolones

in ESBL producers [7, 8, 11] It has also been demonstrated that CTX-M-15 ESBL hydrolyzes cefepime with higher efficiency than other ESBL variation [5] Here, ESBL producers also demonstrated

a high level of resistance against ciprofloxacin, trimethoprim-sulfamethoxazole, nalidixic acid and chloramphenicol Our work shows that ESBL

producing strains carrying blaCTX-M-15 exhibit complete resistance and intermediate resistance to cefepime with

significantly higher MICs than other blaCTX-M alleles This complexity in antimicrobials resistance combinations limits suitable drug of choice for antimicrobial therapy, leaving cabapenems and aminoglycosides the last options for treatment in some cases The emergence of NDM-1 clearly compounds

potential treatment options, and more recent data

additionally suggests an association between

resistance to beta-lactams and aminoglycosides in

ESBL-producing bacteria [22] Furthermore,

organisms carrying ESBLs are highly efficient at

transferring their resistance to other organisms within

the same or different species through conjugation,

increasing the rate of antimicrobial resistance

transmission Selective pressure, from heavy use of

extended-spectrum beta lactam will presumably

maintain the presence of these ESBL-producing

pathogens resulting in the persistence and transmission

of ESBL resistance determinants among

Gram-negative bacteria Therefore, further characterization

of other antimicrobial resistance mechanisms will be

important to understand the co-transmission of a range

of antimicrobial determinants

Our study represents a snap shot of ESBL

producing enteric bacteria causing nosocomial

infections in our setting We report that antimicrobial

resistance in hospital isolates is common in Vietnam

and ESBL organisms are widespread CTX-M-type

ESBLs were the most common enzymes found in both

E coli and K pneumoniae Furthermore, the ESBL

genes were consistently located on highly

transmissible plasmids ranging from 50 to 170 kb in

size We suggest that the rampant use of

extended-spectrum cephalosporins in the hospital is driving the

on-going selection, maintenance and dissemination of

these ESBL genes across a spectrum of Gram-negative

organisms and recommend a stringent review of

antimicrobial use in the Vietnamese healthcare system

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Corresponding author

Pham Thanh Duy

Enteric Infections Group

The Hospital for Tropical Diseases

Wellcome Trust Major Overseas Programme

Oxford University Clinical Research Unit

764 Vo Van Kiet, Quan 5.Ho Chi Minh City, Vietnam

Tel: +84 839 239210 Fax: +84 839 238904

Email: duypt@oucru.org

Conflict of interests:No conflict of interests is declared